The present disclosure relates generally to optically monitoring of cleaving enzyme activity, and more particularly to the optical detection of enzyme (polymerase, DNase, and protease, etc.) activity using a specially designed co-factor labeled protein or oligonucleotide.
Genetic testing and enzyme-based assays have the potential for use in a variety of applications, ranging from genetic diagnostics of human diseases to detection of trace levels of pathogens in food products. Currently, more than 400 diseases can be diagnosed by molecular biology analysis of nucleic acid sequences. It is likely that additional tests will be developed as further genetic information becomes available. DNA diagnostic devices enable clinicians to efficiently detect the presence of a whole array of genetically based diseases, including, for example, AIDS, Alzheimer's, and various forms of cancer.
DNA amplification processes are currently used for real-time and end-point detection of specific DNA sequences. The current chemistries allow detection of PCR products via the generation of a fluorescent signal. Generally, the required reagents for these systems are expensive to synthesize, and in some instances, the systems require use of expensive fluorescence instrumentation for detection. Some techniques include binding dye to a double stranded DNA sequence and thus do not use a probe designed for any particular target being analyzed. However, detection of PCR amplified DNA by such a method requires extensive optimization since the dye cannot distinguish between specific and non-specific products accumulated during PCR. With this type of technique, follow-up assays are used, in some instances, to validate obtained results.
The rising use of DNA and/or protein diagnostic testing devices has produced a need for low-cost, highly portable DNA and/or detection devices (for example, a glucometer-type “lab-on-a-chip” device) for use in various markets including health care, agriculture, food testing and bio-defense. Generally, it would be desirable that any new DNA and/or protein diagnostic devices integrate several functional analysis components within the same platform. Further, it would be desirable that such devices be reliable, inexpensive, and able to simplify the monitoring of PCR (polymerase chain reaction) and EA (cleaving enzyme activity).